The goals of the proposed research are to develop high throughput screens to identify inhibitors specific for the transcriptional function of lysyl-tRNA synthetase (LysRS) in mast activation. By releasing potent pro-inflammatory mediators, mast cell is an essential effectors in the elicitation of allergic responses. Our recent studies indicate that an essential component of the protein synthesis machinery-LysRS --is a key regulator for the production of these mediators. Antigen induction of mast cells triggers the phosphorylation of LysRS on Ser207. In the absence of phosphorylation, LysRS is strongly associated with the cytoplasmic multi-tRNA synthetase complex MSC in a closed form, which catalyzes Lys-tRNALys aminoacylation reaction for protein synthesis. However, phosphorylation of Ser207 triggers LysRS into an open conformer that functions exclusively for transcription. Our results show that, by opening up the structure, phosphorylated LysRS is released from MSC, translocates from cytoplasm to the nucleus, and generates Ap4A to activate the transcription of MITF-targeted genes for mast cell activation. This pathway is the first example of a housekeeping machinery involved in the regulation of mast cell activation. Our central goal is to develop novel screening assays to allow a high throughput screen for specific inhibitors of the LysRS open conformer. As outlined in Aim 1, we will generate a novel HTS-compatible LysRS cell-based assay and, using the resources provided by the MLP initiative and in collaboration with a MLPCN center, will perform a HTS campaign to identify lead LysRS pharmacophores. Once initial 'hits' derived from the MLSMR (molecular libraries small molecule repository) collection are confirmed, a series of HTS-compatible direct screen will be performed to further triage confirmed 'hits' in Aim 2. These secondary screens include a novel protein-protein interaction-based alpha screen assay, a validated cell-based transcription activation assay, and a biochemical activity assay. To rank order compound activity, EC50's will be determined and the chemical tractability of the chose 'hits' will be evaluated. In Aim 3, we will develop a functional cell-based high-content imaging, to quantify cellular potency on the translocation of LysRS, to ensure that the mechanism of action of compounds is indeed due to LysRS inhibition. Finally, using a mast cell line, we will test if our inhibitors affect the mast cell immune activaton (e.g., the expression of MCP6, TPH) and we will rank order of top inhibitors for their ability to modulate transcriptional activation in mast cells. Collectively, our cell-based and biochemical assays, along with profiling lead compounds against a series of mechanistic screens will drive the discovery of LysRS novel and selective molecular probes for its non-canonical function in mast cell activation, with an ultimate goal of developing unique anti-allergy compound.